Novel method for improving solubility of oleosin protein

Abstract

The invention discloses a novel method for improving solubility of oleosin protein, and belongs to the technical field of food. The method disclosed by the invention comprises two steps of oleosin protein extraction and dissolution; the method comprises the following steps: mixing a plant oil body with a non-polar solvent with the volume 3-5 times that of the plant oil body for 15-30 minutes, then centrifuging for 20-30 minutes under the condition of 7000-9000 rpm, adding chloroform / methanol / water (4: 2: 1) with the volume 3-5 times that of the plant oil body into a precipitate, mixing for 10-30 minutes, centrifuging under the condition of 7000-9000 rpm, carrying out nitrogen blowing on the precipitate for 10-30 minutes, freeze-drying for 24-48 hours, adding an ethanol solution with the concentration of 50%-95%, and freeze-drying for 10-48 hours to obtain the plant oil. And mixing for 5 to 15 minutes to obtain an oleosin protein solution. Compared with the prior art, the prepared oleosin protein solution has the advantages that the use of ultrasonic waves, microwaves, urea and other denaturants is avoided, the spatial secondary structure of the protein is kept stable, and the solubility is improved to 0.5 mg / mL to 0.7 mg / mL.

Description

Technical Field

[0001] This invention belongs to the field of lipid and protein processing technology. More specifically, it relates to a novel method for improving the solubility of oleosin protein. Background Technology

[0002] Organelles (OBs) are widely found in plant cells, especially seed cells, and are used to store triglycerides (TAGs) to provide energy for plant life activities. Besides phospholipids, proteins are the most important components of the OB membrane structure, playing a crucial role in maintaining OB stability and endowing them with high tolerance to extreme environments. During OB formation, transmembrane proteins transcribed by ribosomes and embedded in the monolayer phospholipid membrane are called OB intrinsic proteins. OB intrinsic proteins consist of three classes of proteins: oleosin, caleosin, and steroleosin. Among these, oleosin has the highest surface area on the OB membrane and plays the most important role in stabilizing the membrane structure.

[0003] Because it has both hydrophilic and hydrophobic regions, amphiphilic oleosin should theoretically have good interfacial activity and is a potential excellent natural surfactant. Therefore, this natural protein can be used in food, pharmaceuticals and cosmetics. However, the hydrophobic region of oleosin has a very high proportion, resulting in extremely low solubility when water is used as a solvent. The poor solubility of oleosin makes extraction and preparation difficult and also has an adverse effect on the study of its spatial structure and interfacial properties. In order to solve the problem of low solubility and difficult preparation caused by the extremely long transmembrane region of oleosin, the following methods are usually used in the traditional way: (1) Improve the traditional solvent method and use ultrasound-assisted salt dissociation technology to improve the extraction rate and solubility of oleosin. This method can effectively improve the solubility of oleosin, but because the strong shear force caused by ultrasound can destroy the natural conformation of the protein, this method is extremely unfavorable for the study of the spatial conformation of oleosin (CN201811081288.4). (2) Graft dextran onto the non-transmembrane region of oleosin before extraction and preparation. This method also significantly improves its solubility. However, the high temperature of the Maillard reaction may affect the structure and properties of oleosin, and the grafting of dextran changes the structure and interfacial properties of oleosin, which is not conducive to the study of its native conformation and its interaction mechanism with phospholipids and non-intrinsic proteins (CN202211076604.5). (3) Gene cloning technology is used to modify the amino acid sequence of oleosin. By editing the oleosin gene sequence to shorten the hydrophobic water of oleosin, and then using E. coli to express the modified gene sequence, highly soluble recombinant oleosin was obtained. Although this method can successfully prepare high-purity and highly soluble recombinant oleosin, the cost of preparing full-length oleosin by gene cloning technology is high, and oleosin expressed in prokaryotes still needs to be dissolved by adding a high concentration of denaturant, which seriously affects the study of the spatial conformation of oleosin, and at the same time brings new challenges to the subsequent study of its interaction with phospholipids and non-intrinsic proteins. In summary, selecting suitable solvents to maintain the native conformation of oleosin and improve its solubility without using denaturing agents is key to advancing research on the spatial structure of oleosin and its interactions with other molecules.

[0004] Therefore, this invention aims to reduce production costs and shorten the production cycle by adjusting the molecular ratio to change the solution polarity, thereby developing a good solvent for dissolving oleosin protein, optimizing key parameters, and ultimately establishing an efficient method adapted to the dissolution of oleosin protein. Summary of the Invention

[0005] Existing methods for improving oleosin protein solubility, such as using denaturants, ultrasound-assisted salt ionization, and chemical or biological modification techniques, significantly increase the production and usage costs of oleosin protein, increase energy consumption, and cause environmental pollution. Furthermore, these methods require multiple processing steps, resulting in long production and reaction times. Moreover, the improved solubility alters the structure and physicochemical properties of oleosin protein, limiting its application in food, cosmetics, and pharmaceuticals. To address the shortcomings of existing technologies, the applicant, through extensive preliminary research on the physicochemical properties of oleosin protein, creatively discovered that adjusting the molecular ratio to change the solution polarity can improve oleosin protein solubility. The research found that this method of improving oleosin protein solubility by adjusting the molecular ratio to change the solution polarity not only avoids the use of denaturants but also shortens the preparation time and significantly reduces costs compared to ultrasound-assisted salt ionization, chemical, and biological modification techniques.

[0006] This invention addresses the shortcomings of existing technologies by developing a novel, efficient, low-chemical-consumption, low-energy-consumption, and low-cost method for improving oleosin protein solubility. This method is of great significance for improving oleosin protein solubility, conducting in-depth research on the molecular structure and properties of oleosin protein, and broadening the application fields of oleosin protein.

[0007] The purpose of this invention is to provide the application of the novel method for improving the solubility of oleosin protein in the dissolution and processing of oleosin protein.

[0008] To achieve the aforementioned objectives, the present invention employs the following technical solutions: First, the vegetable oil is mixed with a certain volume of nonpolar solvent to remove the neutral lipids contained in the oil. After centrifugation to separate the precipitate from which the neutral lipids have been completely removed, a certain amount of chloroform / methanol / water solution is added and mixed to completely remove the phospholipids. Then, the secondary precipitate is collected by centrifugation. The secondary precipitate is subjected to nitrogen blowing and freeze-drying to obtain the freeze-dried powder of the sample. A certain volume of ethanol solution is added to the freeze-dried powder of the sample and mixed thoroughly to obtain the oleosin protein solution.

[0009] Preferably, the nonpolar solvent is one or more of hexane, diethyl ether, or petroleum ether.

[0010] Preferably, the amount of nonpolar solvent used is 3-5 times the weight of the oil, expressed in g / mL.

[0011] More preferably, the amount of nonpolar solvent used is five times the volume of the oil body, expressed in g / mL.

[0012] Preferably, the mixing time between the oil and the non-polar solvent is 15-30 minutes.

[0013] Preferably, the mixture of oil and non-polar solvent is centrifuged at 7000-9000 rpm.

[0014] Preferably, the centrifugation time for the mixture of oil and non-polar solvent is 20-30 minutes.

[0015] Preferably, the steps of mixing the oil with the non-polar solvent and centrifuging need to be repeated 3-5 times.

[0016] Preferably, the ratio of chloroform / methanol / water is 4:2:1.

[0017] Preferably, the amount of chloroform / methanol / water solution used is 3-5 times the volume of the precipitate mass, expressed in g / L.

[0018] More preferably, the amount of chloroform / methanol / water solution used is five times the volume of the precipitate, expressed in g / L.

[0019] Preferably, the mixing time of the precipitate with chloroform / methanol / water solution is 15-30 minutes.

[0020] Preferably, the centrifugation conditions for the mixture of precipitate and chloroform / methanol / aqueous solution are 7000-9000 rpm.

[0021] Preferably, the centrifugation time of the mixture of precipitate and chloroform / methanol / aqueous solution is 20-30 minutes.

[0022] Preferably, the step of mixing the precipitate with chloroform / methanol / aqueous solution and centrifuging should be repeated 2-3 times.

[0023] Preferably, the secondary precipitate needs to be purged with nitrogen for 10-30 minutes.

[0024] Preferably, the secondary precipitate needs to be freeze-dried for 24-48 hours.

[0025] Preferably, the concentration of the ethanol solution is in the range of 50%-95%.

[0026] Preferably, the pH value of the ethanol solution is in the range of 3-7.

[0027] Preferably, the amount of ethanol solution used, expressed in g / mL, is 0.3-1 times the volume of the lyophilized powder.

[0028] Preferably, the mixing time between the lyophilized powder and the ethanol solution is 5-15 minutes.

[0029] The present invention also provides an oleosin protein solution prepared by the above-described novel method for improving the solubility of oleosin protein.

[0030] Preferably, the oleosin protein solution has a solubility of 0.5 mg / mL–0.7 mg / mL, and the α-helical structure accounts for more than 40% of the secondary structure.

[0031] The present invention also provides the application of the above-mentioned new method for improving the solubility of oleosin protein in the dissolution and processing of oleosin protein.

[0032] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention provides a novel method for improving the solubility of oleosin protein. This method is safe, simple to operate, highly efficient, requires minimal chemical consumption, low energy consumption, and has low production costs. The method alters the solution polarity by adjusting the molecular ratio and makes corresponding modifications to the characteristics of oleosin protein to enhance its solubility. Compared to traditional methods for improving oleosin protein solubility, this method significantly increases the solubility to over 0.5 mg / mL. Furthermore, this method eliminates the need for ultrasonic treatment, simplifies operation, reduces energy consumption, and eliminates the need for denaturing agents such as urea and trichloroacetic acid. It also reduces chemical consumption, significantly shortens processing time, and substantially lowers costs, resulting in high production efficiency and low production costs. Attached Figure Description

[0033] Figure 1 This is a flowchart illustrating a new method for improving the solubility of oleosin protein.

[0034] Figure 2 The solubility diagrams are for the oleosin protein solutions prepared in Examples 1-5 and Comparative Examples 1 and 2.

[0035] Figure 3 The solubility diagram shows the oleosin protein solutions prepared in Examples 6-10.

[0036] Figure 4 The diagram shows the proportion of secondary structures in the oleosin protein solutions prepared in Examples 6-10 under different pH conditions.

[0037] Figure 5 The fluorescence spectra of the oleosin protein solutions prepared in Examples 6-10 under different pH conditions are shown. Detailed Implementation

[0038] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0039] Figure 1 The flowchart of a novel method for improving the solubility of oleosin protein provided by the present invention is shown.

[0040] Example 1 The nonpolar solvent used in this embodiment is diethyl ether, the ethanol solution concentration is 55% (v / v), and the amount of ethanol solution added, in g / mL, is 30% (0.3 times) of the mass of the lyophilized powder. The pH of the ethanol solution used is 7, and the mixing time of the ethanol solution and the lyophilized powder is 5 minutes. A new method for improving the solubility of oleosin protein includes the following steps: Accurately weigh 5g of oil and add anhydrous diethyl ether (3 times the weight of the oil, g / mL) at room temperature (25℃). Mix thoroughly for 15 minutes. Centrifuge the mixture at 9000 rpm for 20 minutes. Repeat this step three times. After separating the precipitate, add a chloroform / methanol / water solution in a ratio of 4:2:1 (3 times the weight of the precipitate, g / L). Mix thoroughly for 15 minutes. Centrifuge the mixture at 7000 rpm for 30 minutes. Repeat this step twice. Expel nitrogen to the secondary precipitate for 30 minutes and then freeze-dry for 48 hours to obtain a lyophilized powder. Accurately weigh 1mg of the lyophilized powder and add 0.3 mL of 55% (v / v) ethanol solution. Mix thoroughly for 5 minutes to obtain an oleosin protein solution.

[0041] Example 2 The nonpolar solvent used in this embodiment is diethyl ether, the ethanol solution concentration is 65% (v / v), and the amount of ethanol solution added, in g / mL, is 80% (0.8 times) of the mass of the lyophilized powder. The pH of the ethanol solution used is 7, and the mixing time of the ethanol solution and the lyophilized powder is 8 minutes. A new method for improving the solubility of oleosin protein includes the following steps: Accurately weigh 5g of oil and add anhydrous diethyl ether (5 times the weight of the oil, g / mL) at room temperature (25℃). Mix thoroughly for 30 minutes. Centrifuge the mixture at 8000 rpm for 30 minutes. Repeat this step 5 times. After separating the precipitate, add a chloroform / methanol / water solution in a ratio of 4:2:1 (5 times the volume of the precipitate, g / L). Mix thoroughly for 20 minutes. Centrifuge the mixture at 8000 rpm for 30 minutes. Repeat this step 3 times. Expel nitrogen to the secondary precipitate for 30 minutes, then freeze-dry for 48 hours to obtain a lyophilized powder. Accurately weigh 1mg of the lyophilized powder and add 0.8 mL of 65% (v / v) ethanol solution. Mix thoroughly for 8 minutes to obtain an oleosin protein solution.

[0042] Example 3 The nonpolar solvent used in this embodiment is diethyl ether, the ethanol solution concentration is 75% (v / v), and the amount of ethanol solution added, in g / mL, is 50% (0.5 times) of the mass of the lyophilized powder. The pH of the ethanol solution used is 7, and the mixing time of the ethanol solution and the lyophilized powder is 10 minutes. A new method for improving the solubility of oleosin protein includes the following steps: Accurately weigh 5g of oil and add anhydrous diethyl ether (5 times the weight of the oil, g / mL) at room temperature (25℃). Mix thoroughly for 30 minutes. Centrifuge the mixture at 7000 rpm for 20 minutes. Repeat this step 5 times. After separating the precipitate, add a chloroform / methanol / water solution in a ratio of 4:2:1 (3 times the volume of the precipitate, g / L). Mix thoroughly for 30 minutes. Centrifuge the mixture at 9000 rpm for 30 minutes. Repeat this step 3 times. Expel nitrogen to the secondary precipitate for 30 minutes, then freeze-dry for 24 hours to obtain a lyophilized powder. Accurately weigh 1mg of the lyophilized powder and add 0.5 mL of 75% (v / v) ethanol solution. Mix thoroughly for 10 minutes to obtain an oleosin protein solution.

[0043] Example 4 The nonpolar solvent used in this embodiment is diethyl ether, the ethanol solution concentration is 85% (v / v), and the amount of ethanol solution added, in g / mL, is 100% (1 times) of the mass of the lyophilized powder. The pH of the ethanol solution used is 7, and the mixing time of the ethanol solution and the lyophilized powder is 15 minutes. A new method for improving the solubility of oleosin protein includes the following steps: Accurately weigh 5g of oil and add anhydrous diethyl ether (5 times the weight of the oil, g / mL) at room temperature (25℃). Mix thoroughly for 30 minutes. Centrifuge the mixture at 7000 rpm for 20 minutes. Repeat this step 5 times. After separating the precipitate, add a chloroform / methanol / water solution in a ratio of 4:2:1 (3 times the volume of the precipitate, g / L). Mix thoroughly for 30 minutes. Centrifuge the mixture at 9000 rpm for 30 minutes. Repeat this step 3 times. Exfoliate the secondary precipitate under nitrogen for 30 minutes, then freeze-dry for 24 hours to obtain a lyophilized powder. Accurately weigh 1mg of the lyophilized powder and add 1mL of 85% (v / v) ethanol solution. Mix thoroughly for 15 minutes to obtain an oleosin protein solution.

[0044] Example 5 The nonpolar solvent used in this embodiment is diethyl ether, the ethanol solution concentration is 95% (v / v), and the amount of ethanol solution added, in g / mL, is 50% (0.5 times) of the mass of the lyophilized powder. The pH of the ethanol solution used is 7, and the mixing time of the ethanol solution and the lyophilized powder is 15 minutes. A new method for improving the solubility of oleosin protein includes the following steps: Accurately weigh 5g of oil and add anhydrous diethyl ether (5 times the weight of the oil, g / mL) at room temperature (25℃). Mix thoroughly for 30 minutes. Centrifuge the mixture at 7000 rpm for 20 minutes. Repeat this step 5 times. After separating the precipitate, add a chloroform / methanol / water solution in a ratio of 4:2:1 (3 times the volume of the precipitate, g / L). Mix thoroughly for 30 minutes. Centrifuge the mixture at 9000 rpm for 30 minutes. Repeat this step 3 times. Exfoliate the secondary precipitate under nitrogen for 30 minutes, then freeze-dry for 24 hours to obtain a lyophilized powder. Accurately weigh 1mg of the lyophilized powder and add 0.5 mL of 95% (v / v) ethanol solution. Mix thoroughly for 15 minutes to obtain an oleosin protein solution.

[0045] Example 6 The nonpolar solvent used in this embodiment is diethyl ether, the ethanol solution concentration is 65% (v / v), and the amount of ethanol solution added, in g / mL, is 100% (1 times) of the mass of the lyophilized powder. The pH of the ethanol solution used is 3, and the mixing time of the ethanol solution and the lyophilized powder is 10 minutes. A new method for improving the solubility of oleosin protein includes the following steps: Accurately weigh 5g of oil and add anhydrous diethyl ether (g / mL, based on the oil weight) at room temperature (25℃). Mix thoroughly for 15 minutes. Centrifuge the mixture at 7000 rpm for 30 minutes. Repeat this step three times. After separating the precipitate, add a chloroform / methanol / water solution in a ratio of 4:2:1 (3 times the volume of the precipitate, g / L). Mix thoroughly for 15 minutes. Centrifuge the mixture at 8000 rpm for 30 minutes. Repeat this step twice. Expel nitrogen to the secondary precipitate for 30 minutes and then freeze-dry for 24 hours to obtain a lyophilized powder. Accurately weigh 1mg of the lyophilized powder and add 1mL of 65% (v / v) ethanol solution. Mix thoroughly for 10 minutes to obtain an oleosin protein solution.

[0046] Example 7 The nonpolar solvent used in this embodiment is diethyl ether, the ethanol solution concentration is 65% (v / v), and the amount of ethanol solution added, in g / mL, is 50% (0.5 times) of the mass of the lyophilized powder. The pH of the ethanol solution used is 4, and the mixing time between the ethanol solution and the lyophilized powder is 5 minutes. A new method for improving the solubility of oleosin protein includes the following steps: Accurately weigh 5g of oil and add 5 times the volume of n-hexane (g / mL, based on oil weight) at room temperature (25℃). Mix thoroughly for 30 minutes. Centrifuge the mixture at 9000 rpm for 20 minutes. Repeat this step 3 times. After separating the precipitate, add a chloroform / methanol / water solution in a ratio of 4:2:1, using 5 times the volume (g / L) of the precipitate. Mix thoroughly for 15 minutes. Centrifuge the mixture at 7000 rpm for 20 minutes. Repeat this step 3 times. Expel nitrogen to the secondary precipitate for 10 minutes, then freeze-dry for 48 hours to obtain a lyophilized powder. Accurately weigh 1mg of the lyophilized powder and add 0.5 mL of 65% (v / v) ethanol solution. Mix thoroughly for 5 minutes to obtain an oleosin protein solution.

[0047] Example 8 The nonpolar solvent used in this embodiment is diethyl ether, the ethanol solution concentration is 65% (v / v), and the amount of ethanol solution added, in g / mL, is 50% (0.5 times) of the mass of the lyophilized powder. The pH of the ethanol solution used is 5, and the mixing time between the ethanol solution and the lyophilized powder is 5 minutes. A new method for improving the solubility of oleosin protein includes the following steps: Accurately weigh 5g of oil and add 5 times the volume of n-hexane (g / mL, based on oil weight) at room temperature (25℃). Mix thoroughly for 30 minutes. Centrifuge the mixture at 9000 rpm for 20 minutes. Repeat this step 3 times. After separating the precipitate, add a chloroform / methanol / water solution in a ratio of 4:2:1, using 5 times the volume (g / L) of the precipitate. Mix thoroughly for 15 minutes. Centrifuge the mixture at 7000 rpm for 20 minutes. Repeat this step 3 times. Expel nitrogen to the secondary precipitate for 10 minutes, then freeze-dry for 48 hours to obtain a lyophilized powder. Accurately weigh 1mg of the lyophilized powder and add 0.5 mL of 65% (v / v) ethanol solution. Mix thoroughly for 5 minutes to obtain an oleosin protein solution.

[0048] Example 9 The nonpolar solvent used in this embodiment is diethyl ether, the ethanol solution concentration is 65% (v / v), and the amount of ethanol solution added, in g / mL, is 50% (0.5 times) of the mass of the lyophilized powder. The pH of the ethanol solution used is 6, and the mixing time between the ethanol solution and the lyophilized powder is 5 minutes. A new method for improving the solubility of oleosin protein includes the following steps: Accurately weigh 5g of oil and add 5 times the volume of n-hexane (g / mL, based on oil weight) at room temperature (25℃). Mix thoroughly for 30 minutes. Centrifuge the mixture at 9000 rpm for 20 minutes. Repeat this step 3 times. After separating the precipitate, add a chloroform / methanol / water solution in a ratio of 4:2:1, using 5 times the volume (g / L) of the precipitate. Mix thoroughly for 15 minutes. Centrifuge the mixture at 7000 rpm for 20 minutes. Repeat this step 3 times. Expel nitrogen to the secondary precipitate for 10 minutes, then freeze-dry for 48 hours to obtain a lyophilized powder. Accurately weigh 1mg of the lyophilized powder and add 0.5 mL of 65% (v / v) ethanol solution. Mix thoroughly for 5 minutes to obtain an oleosin protein solution.

[0049] Example 10 The nonpolar solvent used in this embodiment is diethyl ether, the ethanol solution concentration is 65% (v / v), and the amount of ethanol solution added, in g / mL, is 50% (0.5 times) of the mass of the lyophilized powder. The pH of the ethanol solution used is 7, and the mixing time of the ethanol solution and the lyophilized powder is 10 minutes. A new method for improving the solubility of oleosin protein includes the following steps: Accurately weigh 5g of oil and add anhydrous diethyl ether (5 times the weight of the oil, g / mL) at room temperature (25℃). Mix thoroughly for 30 minutes. Centrifuge the mixture at 9000 rpm for 20 minutes. Repeat this step three times. After separating the precipitate, add a chloroform / methanol / water solution in a ratio of 4:2:1 (3 times the volume of the precipitate, g / L). Mix thoroughly for 20 minutes. Centrifuge the mixture at 8000 rpm for 30 minutes. Repeat this step three times. Exfoliate the secondary precipitate under nitrogen for 30 minutes, then freeze-dry for 48 hours to obtain a lyophilized powder. Accurately weigh 1mg of the lyophilized powder and add 0.5 mL of 65% (v / v) ethanol solution. Mix thoroughly for 10 minutes to obtain an oleosin protein solution.

[0050] Comparative Example 1 The nonpolar solvent used in this comparative example was diethyl ether, and the solvent for dissolving oleosin protein was ultrapure water. Accurately weigh 5g of oil and add anhydrous diethyl ether (5 times the weight of the oil, g / mL) at room temperature (25℃). Mix thoroughly for 30 minutes. Centrifuge the mixture at 9000 rpm for 20 minutes. Repeat this step three times. After separating the precipitate, add a chloroform / methanol / water solution in a ratio of 4:2:1 (3 times the weight of the precipitate, g / L). Mix thoroughly for 20 minutes. Centrifuge the mixture at 8000 rpm for 30 minutes. Repeat this step three times. Expel nitrogen to the secondary precipitate for 30 minutes, then freeze-dry for 48 hours to obtain a lyophilized powder. Accurately weigh 1mg of the lyophilized powder and add 0.5mL of ultrapure water. Mix thoroughly for 5 minutes to obtain an oleosin protein solution.

[0051] Comparative Example 2 The nonpolar solvent used in this comparative example was diethyl ether, and the solvent for dissolving oleosin protein was anhydrous ethanol. Accurately weigh 5g of oil and add anhydrous diethyl ether (5 times the weight of the oil, g / mL) at room temperature (25℃). Mix thoroughly for 30 minutes. Centrifuge the mixture at 9000 rpm for 20 minutes. Repeat this step three times. After separating the precipitate, add a chloroform / methanol / water solution in a ratio of 4:2:1 (3 times the volume of the precipitate, g / L). Mix thoroughly for 20 minutes. Centrifuge the mixture at 8000 rpm for 30 minutes. Repeat this step three times. Expel nitrogen to the secondary precipitate for 30 minutes, then freeze-dry for 48 hours to obtain a lyophilized powder. Accurately weigh 1mg of the lyophilized powder and add 0.5 mL of anhydrous ethanol. Mix thoroughly for 5 minutes to obtain an oleosin protein solution.

[0052] Example Quality Inspection and Result Analysis (1) Determination of the solubility of oleosin protein solutions prepared in Examples 1-5 and Comparative Examples 1-2: The absorbance at 280 nm was measured using a UV spectrophotometer to determine the solubility of the oleosin protein solutions in Examples 1-5 and Comparative Examples 1-2.

[0053] Figure 2 The results show the solubility of oleosin protein in different examples and comparative examples. The results indicate that as the ethanol concentration increased from 55% to 65%, the solubility of oleosin protein gradually increased from 0.4 mg / mL to approximately 0.7 mg / mL; however, as the ethanol concentration further increased, the solubility of oleosin gradually decreased; when the concentration reached 95%, the solubility of oleosin was less than 0.05 mg / mL. (2) Determination of the solubility of oleosin protein solutions prepared in Examples 6-10 at different pH values: The absorbance at 280 nm was measured using a UV spectrophotometer to determine the solubility changes of Examples 6-10 under different pH conditions.

[0054] In Examples 6-10, the solubility of oleosin protein exceeded 0.5 mg / mL under different pH conditions, but the solubility varied significantly with pH: the solubility of oleosin protein was highest at pH=3 and 7, while the solubility was lowest at pH=5. This phenomenon may be related to pH changes inducing conformational changes in the protein, leading to alterations in its aggregation degree. When the aggregation degree changes, the surface charge of the protein decreases, the molecular repulsion decreases, and the solubility decreases.

[0055] (3) Determination of the proportion of secondary structure of oleosin protein solutions prepared in Examples 6-10 at different pH values: The changes in the proportion of secondary structures of oleosin protein with pH in Examples 6-10 were determined using circular dichroism spectroscopy.

[0056] like Figure 3As shown, the α-helix proportion in the secondary structure of the oleosin protein in Example 8 was lowest at pH=5; while the α-helix proportion was highest in the oleosin proteins of Examples 6 and 10 at pH=3 and pH=7. When the oleosin protein is in acidic and neutral environments, the proportion of α-helices in the secondary structure gradually decreases while the proportion of random coil structures increases. The flexibility of the hydrophobic water area in solution increases, and hydrophobic amino acids are more easily exposed to the environment, resulting in enhanced hydrophobic interactions between protein molecules. The proportion of α-helical structure in the secondary structure is higher than 40%.

[0057] (4) Fluorescence spectroscopy determination of oleosin protein solutions prepared in Examples 6-10 at different pH values: The tryptophan and tyrosine residues of oleosin protein are only present in the non-transmembrane domain, so changes in autofluorescence intensity can reflect structural changes in the protein's hydrophilic domain. Figure 4 The results showed that in Examples 6-10, the fluorescence intensity of oleosin protein gradually increased from pH 3 to 5, while gradually decreased from pH 5 to 7. The main reason for this phenomenon is that at pH 5, the hydrophilic arm segments of oleosin exhibit more flexible extension during aggregate formation, making tryptophan and tyrosine more easily exposed to the environment, resulting in the strongest fluorescence peak intensity. Under other pH conditions, the hydrophilic arm structure is more rigid, making it difficult for aromatic amino acids to be exposed to the solvent environment, causing the protein's spatial conformation to mask the fluorescence signals of tryptophan and tyrosine, thus weakening the fluorescence intensity.

[0058] In summary, the present invention has the following beneficial effects: This invention provides a novel method for improving the solubility of oleosin protein. This method is safe, simple to operate, highly efficient, requires minimal chemical consumption, low energy consumption, and has low production costs. The method alters the solution polarity by adjusting the molecular ratio and makes corresponding modifications to the characteristics of oleosin protein to enhance its solubility. Compared to traditional methods for improving oleosin protein solubility, this method significantly increases the solubility to over 0.5 mg / mL. Furthermore, this method eliminates the need for ultrasonic treatment, simplifies operation, reduces energy consumption, and eliminates the need for denaturing agents such as urea and trichloroacetic acid. It also reduces chemical consumption, significantly shortens processing time, and substantially lowers costs, resulting in high production efficiency and low production costs.

[0059] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A novel method for improving the solubility of oleosin protein, characterized in that, Includes the following steps: First, the vegetable oil is mixed with a certain volume of nonpolar solvent to remove the neutral lipids contained in the oil. After centrifugation to separate the precipitate from which the neutral lipids have been completely removed, a certain amount of chloroform / methanol / water solution is added and mixed to completely remove the phospholipids. Then, the secondary precipitate is collected by centrifugation. The secondary precipitate is then subjected to nitrogen blowing and freeze-drying to obtain the freeze-dried sample powder. A certain volume of ethanol solution is added to the freeze-dried sample powder and mixed thoroughly to obtain the oleosin protein solution.

2. A novel method for improving the solubility of oleosin protein according to claim 1, characterized in that, The non-polar solvent is one or more of hexane, diethyl ether, or petroleum ether, and the amount of non-polar solvent used is 3-5 times the weight of the oil, expressed in g / mL.

3. A novel method for improving the solubility of oleosin protein according to claim 1, characterized in that, The mixing time between the non-polar solvent and the oil is 15-30 minutes, and the centrifugation conditions are 7000-9000 rpm for 20-30 minutes. The mixing and centrifugation steps of the oil and non-polar solvent need to be repeated 3-5 times.

4. A novel method for improving the solubility of oleosin protein according to claim 1, characterized in that, The ratio of the three components in the chloroform / methanol / aqueous solution is 4:2:

1. The amount of chloroform / methanol / aqueous solution used is 3-5 times the volume of the precipitate, expressed in g / L. The mixing time between the chloroform / methanol / aqueous solution and the precipitate is 15-30 minutes, and the centrifugation conditions are 7000-9000 rpm for 20-30 minutes. The steps of mixing the precipitate with the chloroform / methanol / aqueous solution and centrifuging are repeated 2-3 times.

5. A novel method for improving the solubility of oleosin protein according to claim 1, characterized in that, The secondary precipitate needs to be purged with nitrogen for 10-30 minutes, and then freeze-dried for 24-48 hours to obtain the freeze-dried powder of the sample.

6. A novel method for improving the solubility of oleosin protein according to claim 1, characterized in that, The volume concentration of the ethanol solution is 55%-95%, and the pH value is in the range of 3-7.

7. A novel method for improving the solubility of oleosin protein according to claim 1, characterized in that, The amount of ethanol solution used, expressed in g / mL, is 0.3-1 times the volume of the lyophilized powder.

8. A novel method for improving the solubility of oleosin protein according to claim 1, characterized in that, The ethanol needs to be thoroughly mixed for 5-15 minutes after being added to the lyophilized powder.

9. An oleosin protein solution prepared by a novel method for improving the solubility of oleosin protein according to any one of claims 1 to 8, characterized in that, The oleosin protein solution exhibits a solubility of 0.5 mg / mL–0.7 mg / mL, with the α-helical structure accounting for more than 40% of the secondary structure.

10. The application of the novel method for improving the solubility of oleosin protein as described in any one of claims 1 to 8 in the dissolution and processing of oleosin protein.

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